Selective recognition, sorting, and localization of proteins to the degradative lysosome via vesicular carriers is an essential feature of all eukaryotic cells. Mislocalization of lysosomal proteases is associated with disease states including lysosomal storage diseases and cancer. Most lysosomal proteases are synthesized in the rough endoplasmic reticulum and transmit through the early secretory pathway en route to the lysosome; they diverge from secreted proteins at a trans-Golgi compartment. One major post-Golgi pathway to the lysosome involves the endosome which is responsible for uncoupling of lysosomal proteases from their receptors. While several genes and gene products have been identified that are involved in the lysosomal delivery of luminal hydrolases at the pre-endosome and endosome stages, post-endosomal events are relatively poorly defined. We propose to characterize post- endosomal steps in lysosomal delivery of luminal hydrolases using the yeast Saccharomyces cerevisiae. The yeast vacuole is functionally equivalent to the mammalian lysosome, and its carboxy peptidase Y (CPY)-pathway of vacuolar delivery parallels the mannose-6-phosphate pathway in mammalian cells. Yeast offers the advantage of well- developed genetics and molecular genetics. The objective of our studies is to define genes and gene products involved in the endosome to lysosome stage of targeting and delivery of luminal proteases that utilize the CPY-pathway. We have developed a novel mutant screen based on immunodetection of a maturation intermediate (p2CPY) of the vacuolar protease carboxypeptidase Y (mCPY). We have isolated mutants that exhibit internal p2CPY accumulation. The mutants display characteristics that indicate defects in endosome to vacuole delivery and process (env mutants). Our specific aims are to characterize four env mutants genetically, microscopically and biochemically; to clone their putative ENV genes, and to begin molecular characterization of the cloned genes and their products.